Phage Display to Identify Peptides Binding to or Penetrating the Mouse Zona Pellucida
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The objective of this study was to identify peptide ligands, using phage display techniques, which bind sites on mouse embryos, ovaries, cytoplasmic membranes and/or intracytoplasmic components. Specifically, M13 coliphage 7-mer, 12-mer and 15-mer random peptide libraries were used separately for biopanning. Peptides derived from the amplified pools were sequenced and studied. The phage display for in vivo ovary experiments yielded no pool of peptides after two cycles of biopanning and re-amplification. With the same initial concentration of a random 7-mer or 12-mer library, there were repeating sequences derived after three and four biopanning cycles on mouse embryos and unfertilized ova. The sequences were not distinguishable from a control group. Subsequent experimentation using a random 15-mer library to select for internalized phage-peptides yielded two apparent consensus sequences, RNVPPIFNDVYWIAF (9/32 or 28%) and HGRFILPWWYAFSPS (11/32 or 34%). The 15-mer control group yielded no clones. The deduced peptide sequences were compared to known sequences to ascertain their uniqueness. No significant similarities were found, yielding two possible novel motifs. Through this adapted process of phage display and further research, the phage display technology may be used as a tool in the recognition of specific mouse gamete sites. By identifying binding sites of mouse gametes, the peptides might be exploited as a means of studying the embryo cell surface or cytoplasmic components and mouse sperm-egg interactions. Such peptides may also be used for macromolecule delivery in transfection or transgenesis.
- Masters Theses